CN106434661A - Regulatory factor for cells under holding state of pseudomonas aeruginosa and application thereof - Google Patents
Regulatory factor for cells under holding state of pseudomonas aeruginosa and application thereof Download PDFInfo
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Abstract
The invention relates to a regulatory factor for cells under holding state of pseudomonas aeruginosa and an application thereof. The regulatory factor for cells under holding state of pseudomonas aeruginosa is a micro-molecular single-stranded DNA; the nucleotide sequence is as shown in SEQ ID NO.1 in the sequence list; the whole length is 12bp. The nucleotide sequence of a binding protein related to bacteria surface and used for adjusting the switching between the cells under holding state of pseudomonas aeruginosa and the normal cells is as shown in SEQ ID NO.2. A molecule component composed of the regulatory factor for cells under holding state of pseudomonas aeruginosa and the binding protein related to bacteria surface is capable of effectively regulating the switching between the cells under holding state of pseudomonas aeruginosa and the normal cells and supplying a related targeted locus for the drug for eliminating the cells under holding state of pseudomonas aeruginosa.
Description
Technical field
The present invention relates to a kind of Pseudomonas aeruginosa is held, and the state cell regulate and control factor and its application is stayed, belong to biological medicine technology
Field.
Background technology
Hold stay bacterium be certain bacterial community in the case of lethasl concentration Antibacterial pressure, different with the random phenotype of certain proportion
The little subgroup that changes.Show as temporary sleep state or slow growth conditions, the antibacterials of tolerable lethasl concentration, but this anti-
Bacterium Drug tolerance can not heredity.After the distress resolves of lethasl concentration antibacterials, hold and stay bacterium revert to rapidly again normally
Cell, to antibacterial medicaments insensitive.Bacterium is stayed to have height endurability to antibacterials due to holding, bacterium antimicrobial drug in vivo is stayed in holding for remaining
Infection can be caused again when the reduction of thing concentration or immunity degradation.Thus removing to hold stays bacterium to infect with weight for effective control
Want meaning.With the intersection of subject and going deep into for research, hold and bacterium is stayed gradually by Medical Microbiology, environmental microbiology and micro- life
Each area research person is of interest for bioecology etc., and different holding stays bacterium Forming Mechanism constantly to be found.
Toxin-antitoxin (Toxin Antitoxin) system (TA system) is to study more clearly to hold at present to stay bacterium shape
Become mechanism, it can change some main vital movement states of cell, suppress the synthesis of some macromole, so that at cell
State is stayed in holding.Additionally, can induce cell formed hold stay the factor of state also include DNA damage, high temperature and acid fracturing power, respiration inhibition,
Chemical signal response etc..Research finds, holding for different bacterium stays bacterium Forming Mechanism to be not quite similar, and holding for antibacterial of the same race stays bacterium
Forming Mechanism is also non-single.By the research that deepens continuously of each area research scholar, difference is held and stays bacterium Forming Mechanism to be found.But
It is not yet to find at present fully erased to hold the antibacterials or other means for staying bacterium, it is seen that effective control is held and stays bacterium infection face
Face baptism.
Content of the invention
The technical problem to be solved is to provide a kind of Pseudomonas aeruginosa and hold to stay the state cell regulate and control factor.
Present invention technical problem also to be solved be provide a kind of Pseudomonas aeruginosa hold stay the state cell regulate and control factor should
With.
The technical problem that the present invention is finally solved is to provide to hold with above-mentioned Pseudomonas aeruginosa to stay the state cell regulate and control factor to tie
The bacterium surface associated protein of conjunction and its application.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows：
A kind of Pseudomonas aeruginosa is held and stays the state cell regulate and control factor, described Pseudomonas aeruginosa hold stay state cell regulate and control because
Son is small molecule single stranded DNA, and in nucleotide sequence such as sequence table shown in SEQ ID NO.1, total length is 12bp.
Described Pseudomonas aeruginosa is held and stays the state cell regulate and control factor preferably 5 ' terminal phosphates or the non-phosphorylation in 5 ' ends；
The Pseudomonas aeruginosa of 5 ' terminal phosphates is held and stays the state cell regulate and control factor to be referred to as P poly (C)；5 ' ends are unphosphorylated
Pseudomonas aeruginosa is held and stays the state cell regulate and control factor to be referred to as poly (C).
poly(C)：5’‐CCCCCCCCCCCC‐3’
P‐poly(C)：PO4 3‐5’‐CCCCCCCCCCCC‐3’
Pseudomonas aeruginosa of the present invention is held and stays the state cell regulate and control factor to hold and stay state thin in control Pseudomonas aeruginosa
Application in switching between born of the same parents and normal cell.
Application of the present invention, the Pseudomonas aeruginosa of preferably 5 ' terminal phosphates is held and stays the state cell regulate and control factor and copper
Green pseudomonass bacterium surface Binding proteins are combined, and Pseudomonas aeruginosa will be changed into hold by normal cell and stay state thin
Born of the same parents；Holding when the unphosphorylated Pseudomonas aeruginosa in 5 ' ends stays the state cell regulate and control factor related to Pseudomonas aeruginosa bacteria surface
When associated proteins are combined, Pseudomonas aeruginosa can stay state cell to be changed into normal cell from holding；Described Pseudomonas aeruginosa bacteria
Surface Binding proteins aminoacid sequence contains 352 aminoacid as shown in SEQ ID NO.2, altogether.
A kind of adjust Pseudomonas aeruginosa and hold stay the bacterium surface for switching between state cell and normal cell related to combine egg
In vain, its aminoacid sequence is as shown in SEQ ID NO.2.
The gene of the described bacterium surface Binding proteins of coding, its nucleotide sequence, should as shown in SEQ ID NO.3
It is 65.72% that full length gene is 1059bp, G+C content.
Described bacterium surface Binding proteins as therapy target control Pseudomonas aeruginosa hold stay state cell with
Between normal cell switch in application, the Pseudomonas aeruginosa of 5 ' described terminal phosphates hold stay the state cell regulate and control factor with
Described Pseudomonas aeruginosa bacteria surface Binding proteins are combined or the related knot in described Pseudomonas aeruginosa bacteria surface
During hop protein zero load, Pseudomonas aeruginosa will be changed into hold by normal cell and stay state cell；When the non-phosphoric acid in described 5 ' ends
The Pseudomonas aeruginosa of change is held and stays the state cell regulate and control factor with described Pseudomonas aeruginosa bacteria surface Binding proteins knot
During conjunction, Pseudomonas aeruginosa can stay state cell to be changed into normal cell from holding；The related knot in described Pseudomonas aeruginosa bacteria surface
Hop protein aminoacid sequence is as shown in SEQ ID NO.2.
A kind of regulation and control Pseudomonas aeruginosa is held and stays the reagent for switching between state cell and normal cell, including described Aerugo
Pseudomonass are held and stay the state cell regulate and control factor.
Of the present invention application is all not related to the Therapeutic Method of disease, only limit the use of beyond the non-diseases Therapeutic Method should
With such as in terms of scientific research.
Beneficial effect：
1. the extracellular small molecule single-stranded DNA banks of Pseudomonas aeruginosa are built, screens the Pseudomonas aeruginosa that is effectively controlled
Hold and stay state cell to hold with the Pseudomonas aeruginosa that normal cell mutually switches to stay the state cell regulate and control factor.
2. by two dimensional gel electrophore- sises, Pseudomonas aeruginosa surface Binding proteins are obtained, with above-mentioned Pseudomonas aeruginosa
Hold and stay the state cell regulate and control factor to constitute to hold and stay bacterium regulatory molecule component, effective control Pseudomonas aeruginosa hold stay state cell with normal
The mutual switching of cell.
3. the Pseudomonas aeruginosa surface Binding proteins totally 352 aminoacid, by 1059bp (from start codon to
Termination codon) coding form, its G+C content be 65.72%.
4. the group of molecules for staying the state cell regulate and control factor to constitute with bacterium surface Binding proteins is held by Pseudomonas aeruginosa
Part, can hold, with Effective Regulation Pseudomonas aeruginosa, the switching that stays between state cell and normal cell, be that medicine eliminates the false list of Aerugo
Born of the same parents bacterium is held and stays state cell to provide related target site.
5. held based on Pseudomonas aeruginosa and stay 5 ' terminal phosphate of state cell regulate and control factor single stranded DNA/dephosphorylized to hold
Stay state fast switch over method be directly used in prepare relative medicine and eliminate Pseudomonas aeruginosa and hold and stay state cell.
Description of the drawings
The molecular assemblies regulation and control Aerugo that the extracellular small molecule single stranded DNA of Fig. 1 is constituted with bacterium surface Binding proteins is false single
Born of the same parents bacterium holds the design sketch for staying state cell mutually to switch with normal cell.
Extracellular 5 ' the terminal phosphates of small molecule single stranded DNA of Fig. 2/dephosphorylation regulation and control Pseudomonas aeruginosa is held and stays state cell
The design sketch for mutually switching with normal cell.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and the present invention is merely to illustrate, and should not be also without limitation on sheet described in detail in claims
Invention.
Embodiment 1：The acquisition of the extracellular small molecule single stranded DNA of Pseudomonas aeruginosa
1.1. cultivate to the Pseudomonas aeruginosa bacterium solution centrifugation of stable phase (culture about 33h) and obtain supernatant, supernatant warp
0.22 μm of teflon membrane filter (purchased from Whatman) is filtered and removes remaining thalline.
1.2 process 2h to go through E.C. 3.4.21.64 (protein K, purchased from precious biological engineering (Dalian) company limited) at 37 DEG C
Except contained protein in supernatant.
1.3 above-mentioned obtained products are centrifuged through ultra-filtration centrifuge tube (purchased from Millipore company) and remove macromole double-strand
DNA and remaining macro-molecular protein, obtain the percolate after ultrafiltration.
Percolate after 1.4 ultrafiltration through Deoxydization nucleotide streptokinase-streptodornase (Exonuclease III, (big purchased from precious biological engineering
Even) company limited) and RNase (RNase, purchased from precious biological engineering (Dalian) company limited) in 37 DEG C of process 1h, thus remove
Remaining double-stranded DNA and RNA in percolate.
1.5 obtain the extracellular small molecule single stranded DNA of Pseudomonas aeruginosa using the method for phenol chloroform and ethanol precipitation,
Which is dissolved in sterilized water with the concentration of 20ng/ul.
Embodiment 2：The extracellular small molecule single-stranded DNA banks of Pseudomonas aeruginosa build, and obtain to hold and stay bacterium regulatory factor
The extracellular small molecule single stranded DNA of the Pseudomonas aeruginosa of 2.1 above-mentioned gained is in Deoxydization nucleotide terminal transferase
Under (Terminal Deoxynucleotidyl Transferase, purchased from precious biological engineering (Dalian) company limited) effect, make
Its 3 ' end adds poly (A).
2.2 with oligo (dT) as primer, and using Ex Taq archaeal dna polymerase, (Ex Taq DNA polymerase, is purchased from
Precious biological engineering (Dalian) company limited) complementary strand of above-mentioned fragment is obtained by way of PCR, so as to obtain double-stranded DNA piece
Section.
2.3 by above-mentioned double chain DNA fragment and 19 carrier T of pMD (purchased from precious biological engineering (Dalian) company limited) 16
Under the conditions of DEG C, overnight enzyme joins, and enzyme co-product is reclaimed through PCR cleaning QIAquick Gel Extraction Kit (purchased from Axygen company) cleaning, recovery
DNA is dissolved in the Tris HCl (pH8.0) of 10mmol/L.
2.4 preparation escherichia coli DH, 5 α efficient electric turns competent cell, and concrete grammar is with reference to volumes such as J. Pehanorm Brookers
《Molecular Cloning:A Laboratory guide》.
The enzyme co-product that 2.5 cleanings are reclaimed proceeds to 5 α competent cell of escherichia coli DH in the way of electricity conversion, concrete side
Method is with reference to volumes such as J. Pehanorm Brookers《Molecular Cloning:A Laboratory guide》.LB flat board of the coating containing 100mg/L ampicillin.
After 37 DEG C of culture 24h, obtained clone is isolated and purified.
The nucleotide sequence of 2.6 Insert Fragments carries out sequencing by precious biological engineering (Dalian) company limited.
2.7 Invitrogens (Shanghai) trade Co., Ltd synthesizes the nucleotide sequence obtained by each clone, and tests
Stating a plurality of extracellular small molecule single stranded DNA for being obtained Pseudomonas aeruginosa is held state cell is stayed with having that normal cell mutually switches
Effect property, stays the state cell regulate and control factor so as to obtaining Pseudomonas aeruginosa and holding.Screening Pseudomonas aeruginosa hold stay state cell regulate and control because
The method of son is in the Pseudomonas aeruginosa for having washed extracellular for gained single-stranded small molecule DNA add-back, tests which and holds and stays bacterium ratio.
If treatment group is held and stays bacterium ratio and Pseudomonas aeruginosa not to wash original bacterium solution and hold to stay bacterium ratio identical, we both can determine that at this
The extracellular single-stranded small molecule DNA added by reason group is held for Pseudomonas aeruginosa and stays the state cell regulate and control factor.False single through screening Aerugo
Born of the same parents bacterium is held and stays state cell regulate and control factor nucleotides sequence to be classified as SEQ ID NO.1.
Embodiment 3：The acquisition of Pseudomonas aeruginosa bacteria surface Binding proteins
In 3.1 Invitrogens (Shanghai) trade Co., Ltd synthesizing ribonucleotide sequence such as sequence table shown in SEQ ID NO.1
3 ' ends have the Pseudomonas aeruginosa of Cy5 fluorophor labelling holds and stays bacterium controlling elements.
The 3.2 above-mentioned synthetic products Pseudomonas aeruginosa resuspended with equal-volume phosphate buffer (10mmol/L, pH7.0)
(twice of washing) 37 DEG C of effect 20min.
After 3.3 above-mentioned effect product centrifugations, thalline is washed to remove unnecessary fluorescent labeling regulatory factor.
The resuspended above-mentioned thalline of 3.4 equal-volume phosphate buffers (10mmol/L, pH7.0), using final concentration of 1% first
Aldehyde is crosslinked 15min, then terminates crosslinking 5min with final concentration of 0.125mol/L Glycine.
3.5 above-mentioned products are resuspended in equal-volume phosphate buffer (10mmol/L, pH7.0), ultrasonication.Sink after centrifugation
Forming sediment and the phosphate buffer (10mmol/L, pH7.0) of X containing 2%Triton 100 is dissolved in the concentration of 10 μ g/ μ l, is us
The Pseudomonas aeruginosa cell film total protein for being obtained.
3.6 Pseudomonas aeruginosa cell film total proteins run two-dimensional electrophoresis, and protein is divided each other by isoelectric point, IP and molecular weight
Open.Through fluorescence gel scanner uni silver staining mode, we obtain Pseudomonas aeruginosa bacteria surface Binding proteins.Its aminoacid
Sequence is SEQ ID NO.2, encodes the protein-bonded gene nucleotide series for SEQ ID NO.3.
Embodiment 4：Pseudomonas aeruginosa is formed and holds the functional study for staying state correlation molecule component
4.1 cultivate to stable phase (about 33h) Pseudomonas aeruginosa bacterium solution centrifugation abandon supernatant, thalline is through the abundant water of sterilized water
Wash 2 times.The resuspended thalline of equal-volume LB.
4.2 add final concentration of 5mg/L ofloxacin, 37 DEG C, 250rpm effect 3h by 4.1 bacterium solution for being obtained.Dilution
Coating LB flat board, stays state cell proportion so as to obtaining Pseudomonas aeruginosa under the process (i.e. washing bacterium in Fig. 1) and holding.Right
It is the original Pseudomonas aeruginosa bacterium solution (i.e. original bacteria in Fig. 1) for not removing supernatant according to group.
4.3 by 4.1 bacterium solution for being obtained, and add the poly (C) of final concentration of 10 μm of ol/L, after room temperature effect 10min,
Final concentration of 5mg/L ofloxacin, 37 DEG C, 250rpm effect 3h.Dilution spread LB flat board, so as to obtain the process (i.e. in Fig. 1
Washing bacterium+poly (C)) under Pseudomonas aeruginosa hold and stay state cell proportion.
4.4, by 4.1 bacterium solution for being obtained, add the P poly (C) of final concentration of 10 μm of ol/L, and room temperature acts on 10min
Afterwards, final concentration of 5mg/L ofloxacin, 37 DEG C, 250rpm effect 3h are added.Dilution spread LB flat board, so as to obtain the process
Under (i.e. washing bacterium+P poly (C) in Fig. 1), Pseudomonas aeruginosa is held and stays state cell proportion.
As a result as shown in figure 1, when Pseudomonas aeruginosa bacteria surface Binding proteins are unloaded or with 5 ' terminal phosphates
P poly (C) when combining, Pseudomonas aeruginosa will be changed into hold by normal cell and stay state cell, now hold and stay bacterium ratio relatively
High；When cell surface associated protein and 5 ' ends unphosphorylated poly (C) are combined, Pseudomonas aeruginosa can stay state thin by holding
Born of the same parents are changed into normal cell, now hold and stay bacterium ratio relatively low.
Embodiment 5：Extracellular 5 ' the terminal phosphate of small molecule single stranded DNA of Pseudomonas aeruginosa/dephosphorylized holding stays state fast
Fast handoff functionality research
5.1 cultivate Pseudomonas aeruginosa bacterium solution to stable phase (about 33h) through T4 polynueleotide kinase
(T4polynucleotide kinase, purchased from precious biological engineering (Dalian) company limited) 37 DEG C of process 30min, that is, pass through T4
Polynueleotide kinase effect causes 5 ' terminal phosphate of DNA.It is subsequently adding final concentration of 5mg/L ofloxacin, 37 DEG C,
250rpm acts on 3h.Dilution spread LB flat board, stays state cell proportion (i.e. so as to obtaining Pseudomonas aeruginosa under the process and holding
Original bacteria+phosphorylase in Fig. 2).Matched group is original Pseudomonas aeruginosa bacterium solution (i.e. original bacteria in Fig. 2).
5.2, by 4.1 bacterium solution for being obtained, are separately added into the poly (C) and P poly (C) of final concentration of 10 μm of ol/L,
After room temperature effect 10min, through T4 polynueleotide kinase (T4 polynucleotide kinase, (big purchased from precious biological engineering
Even) company limited) and alkaline phosphatase (alkaline phosphatase (calf intestine), purchased from precious biological work
Journey (Dalian) company limited) process 30min respectively under the conditions of 37 DEG C after, add final concentration of 5mg/L ofloxacin, 37 DEG C,
250rpm acts on 3h.Dilution spread LB flat board, so as to obtain two kinds of process (i.e. washing bacterium+poly (C)+phosphorylase in Fig. 2
Group and washing bacterium+P poly (C)+phosphorylase) under Pseudomonas aeruginosa hold and stay state cell proportion.
As a result as shown in Fig. 2 holding extracellular small molecule in the correlation molecule component for stay state when regulation and control Pseudomonas aeruginosa is formed
When DNA5 ' contains phosphate group in end, Pseudomonas aeruginosa will be changed into hold by normal cell and stay state cell, now hold and stay bacterium
Ratio is higher；When in component, 5 ' end of extracellular small molecule DNA does not have phosphate group, Pseudomonas aeruginosa can stay state cell by holding
It is changed into normal cell, now holds and stay bacterium ratio relatively low.
Claims (8)
1. a kind of Pseudomonas aeruginosa is held and stays the state cell regulate and control factor, it is characterised in that described Pseudomonas aeruginosa is held and stays state thin
Born of the same parents' regulatory factor is small molecule single stranded DNA, in nucleotide sequence such as sequence table shown in SEQ ID NO.1.
2. Pseudomonas aeruginosa according to claim 1 is held and stays the state cell regulate and control factor, it is characterised in that described Aerugo
Pseudomonass are held and stay 5 ' terminal phosphate of the state cell regulate and control factor or the non-phosphorylation in 5 ' ends.
3. the Pseudomonas aeruginosa described in claim 1 or 2 is held and stays the state cell regulate and control factor to hold and stay in control Pseudomonas aeruginosa
Application in switching between state cell and normal cell.
4. application according to claim 3, it is characterised in that the Pseudomonas aeruginosa of 5 ' terminal phosphates is held and stays state cell
Regulatory factor is combined with Pseudomonas aeruginosa bacteria surface Binding proteins, and Pseudomonas aeruginosa will be changed by normal cell
State cell is stayed for holding；Holding when the unphosphorylated Pseudomonas aeruginosa in 5 ' ends stays the state cell regulate and control factor thin with Pseudomonas aeruginosa
When bacterium surface Binding proteins are combined, Pseudomonas aeruginosa can stay state cell to be changed into normal cell from holding；Described Aerugo vacation
Aeruginosa bacteria surface Binding proteins aminoacid sequence is as shown in SEQ ID NO.2.
5. a kind of Pseudomonas aeruginosa that adjusts holds the bacterium surface Binding proteins for staying switching between state cell and normal cell,
It is characterized in that its aminoacid sequence is as shown in SEQ ID NO.2.
6. the gene of the bacterium surface Binding proteins described in claim 5 is encoded, it is characterised in that its nucleotide sequence is such as
Shown in SEQ ID NO.3.
7. the bacterium surface Binding proteins described in claim 5 are held in control Pseudomonas aeruginosa as therapy target and stay state
Application in switching between cell and normal cell, it is characterised in that the Aerugo of 5 ' terminal phosphates described in claim 2
Pseudomonass are held and stay the state cell regulate and control factor and Pseudomonas aeruginosa bacteria surface Binding proteins knot described in claim 5
Close or claim 5 described in the zero load of Pseudomonas aeruginosa bacteria surface Binding proteins when, Pseudomonas aeruginosa will be by
Normal cell is changed into hold and stays state cell；The unphosphorylated Pseudomonas aeruginosa in 5 ' ends described in the claim 2 is held and is stayed
When the state cell regulate and control factor is combined with the Pseudomonas aeruginosa bacteria surface Binding proteins described in claim 5, Aerugo vacation
Zymomonas mobiliss can stay state cell to be changed into normal cell from holding；Described Pseudomonas aeruginosa bacteria surface Binding proteins aminoacid
Sequence is as shown in SEQ ID NO.2.
8. a kind of regulation and control Pseudomonas aeruginosa is held and stays the reagent for switching between state cell and normal cell, it is characterised in that including power
Profit requires that the Pseudomonas aeruginosa described in 1 or 2 is held and stays the state cell regulate and control factor.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20080242627A1 (en) * | 2000-08-02 | 2008-10-02 | University Of Southern California | Novel rna interference methods using dna-rna duplex constructs |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080242627A1 (en) * | 2000-08-02 | 2008-10-02 | University Of Southern California | Novel rna interference methods using dna-rna duplex constructs |
Non-Patent Citations (2)
| Title |
|---|
| RUBENFIELD,M.J.,等: "GP336972.1", 《GENBANK》 * |
| 陈齐，等: "铜绿假单胞菌生物被膜形成及其与持留菌关系研究进展", 《世界临床药物》 * |
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